A Novel Oncolytic Herpes Simplex Virus that Synergizes ... · Cancer Therapy: Preclinical A Novel Oncolytic Herpes Simplex Virus that Synergizes with Phosphoinositide 3-kinase/Akt

Cancer Therapy: Preclinical

A Novel Oncolytic Herpes Simplex Virus that Synergizes withPhosphoinositide 3-kinase/Akt Pathway Inhibitors toTarget Glioblastoma Stem Cells

Ryuichi Kanai, Hiroaki Wakimoto, Robert L. Martuza, and Samuel D. Rabkin

AbstractPurpose: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that

will be effective in glioblastoma stem cells (GSC), an important and untargeted component of GBM. One

approach to enhance oHSV efficacy is by combination with other therapeutic modalities.

Experimental Design: MG18L, containing a US3 deletion and an inactivating LacZ insertion in UL39,

was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection inHSV-

susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with

other oHSVs, alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002,

triciribine, GDC-0941, and BEZ235). Cytotoxic interactions betweenMG18L and PI3K/Akt inhibitors were

determined using Chou–Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant

mouse model of GSC-derived GBM.

Results: MG18L was severely neuroattenuated in mice, replicated well in GSCs, and had anti-GBM

activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs,

whereas their combination with MG18L synergized in killing GSCs and glioma cell lines, but not human

astrocytes, through enhanced induction of apoptosis. Importantly, synergy was independent of inhibitor

sensitivity. In vivo, the combination of MG18L and LY294002 significantly prolonged survival of mice, as

compared with either agent alone, achieving 50% long-term survival in GBM-bearing mice.

Conclusions: This study establishes a novel therapeutic strategy: oHSV manipulation of critical

oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent

for the treatment of GBM, being especially effective when combined with PI3K/Akt pathway–targeted

agents. Clin Cancer Res; 17(11); 3686–96. �2011 AACR.

Introduction

Glioblastoma (GBM) is the most common and deadlyprimary brain tumor in adults (1). Despite advances indrug development, overall survival has not substantiallyimproved. Recently, cancer stem or tumor-initiating cellshave been isolated from solid tumors, including GBM, thatare tumorigenic with characteristics of adult stem cellsincluding self-renewal and differentiation into multiplelineages (2). Glioblastoma stem cells (GSC) are thoughtto be important in GBM progression, heterogeneity, recur-rence, and resistance to therapy and provide an importanttarget for the development of new therapies (3). Detailed

molecular analysis of GBMs has shown that genetic altera-tions in the phosphoinositide-3-kinase (PI3K)/Akt path-way, including in PTEN and PIK3CA, occur in about 80%of tumors (4, 5). These molecular changes confer GBMswith proliferative and survival advantages and may play arole in regulating GSCs (6). There has been rapid growth inthe development and clinical testing of small moleculeinhibitors of the PI3K/Akt pathway including for glioma(7). It has been reported that GSCs are sensitive to Aktinhibitors (8, 9). Unfortunately, the combination ofgenetic alterations and the number of cross-regulatoryfeedback loops in the PI3K/Akt pathway raise questionsabout the likely clinical efficacy of inhibitors alone (7, 10).

Oncolytic viruses are a distinct class of targeted antic-ancer agents with unique mechanisms of action comparedwith other cancer therapies; they selectively replicate in andkill cancer cells (oncolysis), amplifying themselves andspreading but sparing normal tissue. The safety of oncolyticherpes simplex virus (oHSV) therapy has been shown inclinical trials for GBM; however, efficacy remains anecdotaland needs improvement (11, 12). Combining oHSV withchemotherapeutic drugs is an effective strategy to improveoverall efficacy and depending on the specific oHSV muta-tions and drug, synergistic cancer cell killing can be

Authors' Affiliation: Brain Tumor Research Center, Department of Neu-rosurgery, Massachusetts General Hospital and Harvard Medical School,Boston, Massachusetts

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Samuel D. Rabkin, Brain Tumor Research Center,MGH, 185 Cambridge St., CPZN-3800, Boston, MA 02114. Phone: 617-726-6817; Fax: 617-643-3422; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-10-3142

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 17(11) June 1, 20113686

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observed (13). All oHSVvectors developed for clinical use inthebrain contain deletions of the g34.5 gene, themajor viralneurovirulence factor, which is associated with attenuatedreplication, even in cancer cells, and an inability to replicatein GSCs (14, 15). To overcome these obstacles and targetdifferent tumorigenic pathways, we have developed a newclass of oHSV vectors for the treatment of tumors in thebrain that contain a deletion in the HSV-1 US3 gene. US3encodes a serine-threonine kinase that among its manyactivities includes inhibition of virus-induced apoptosisand Akt activation (16–18). As apoptotic pathways arefrequently dysfunctional in tumor cells, US3 mutantsshould engender tumor selectivitybasedonenhancedapop-tosis in normal cells, preventing further replication, as wasfound with R7041 (US3

�) infected human umbilical veinendothelial cell (HUVEC; ref. 18). We previously reportedthat R7041 (i) was efficacious in vitro against a number ofcancer cell lines, including glioma U87 and T98G, and invivo against U87 subcutaneous tumors; (ii) synergized withPI3K/Akt inhibitors; and (iii)was safe after systemic deliveryin the periphery (18). To extend these findings to humanGSCs and intracerebral GBM tumor models for possibleclinical translation, we constructed a new multimutatedoHSV, MG18L [US3 deleted and UL39 (ICP6) negative],which is safe after intracerebral inoculation, replicates wellin GSCs, and synergizes with PI3K/Akt pathway inhibitorsin killing GSCs in vitro and in vivo through enhancedapoptosis.

Materials and Methods

Cell lines and reagentsU87 and T98G human glioma and Vero (African green

monkey kidney) cells were obtained from the American

Type Culture Collection and used at low-passage number.Human astrocytes were obtained from ScienCell. Cellswere maintained in Dulbecco’s modified Eagle’s mediumsupplemented with 10% fetal calf serum (DMEM-FCS) at37�C and 5% CO2. Human GSCs were isolated as pre-viously described (15) and cultured in EF20 mediumcomposed of Neurobasal medium (Invitrogen) supple-mented with 3 mmol/L L-Glutamine (Mediatech), 1�B27 supplement (Invitrogen), 0.5� N2 supplement (Invi-trogen), 2 mg/mL heparin (Sigma), 20 ng/mL human EGF(epidermal growth factor; R&D systems), 20 ng/mL humanFGF-2 (fibroblast growth factor 2; Peprotech) and 0.5�penicillin G/streptomycin sulfate/amphotericin B complex(Mediatech). The stem cell features of GBM4, GBM8, andBT74 have been previously described (15). Spheres weredissociated using NeuroCult Chemical Dissociation kit(StemCell Technologies). Passaged cells were confirmedto be mycoplasma free. LY294002 (LC Laboratories), tri-ciribine (Akt inhibitor V; Santa Cruz Biotechnology), GDC-0941 (Chemdea), BEZ235 (Chemdea), and Z-VAD-FMK(Tocris Bioscience) were dissolved in dimethyl sulfoxide(Sigma-Aldrich).

VirusesAll viruses were constructed on a HSV-1 strain F back-

ground. G207 (g34.5D, ICP6�, LacZ), G47D (g34.5D,ICP6�, ICP47/ US11proD, LacZ), and FD6 (ICP6�, LacZ)have been previously described (14, 15, 19). R7041 (US3deleted) was provided by Dr. B. Roizman (University ofChicago, Chicago, IL; ref. 20). Viruses were grown, purified,and titered on Vero cells (19).

Construction of MG18LConstruction and characterization of MG18L was as

described (19). Briefly, the 5.3-kb fragment of pKX2-bG3(from S.K.Weller, University of Connecticut Health Center,Farmington, CT), containing the Escherichia coli lacZsequence inserted in-frame in UL39, was cotransfected withR7041 viral DNA into Vero cells using Lipofectamine(Invitrogen). Recombinant viruses, isolated by limitingdilution and identified as plaques staining blue after X-gal histochemistry (Supplementary Fig. S2A), were plaquepurified 3 times in Vero cells. The genomic structure ofMG18L was confirmed by restriction endonuclease diges-tion and Southern blot analysis (Supplementary Fig. S1).

Viral replication assayCells were seeded into 24-well plates (2� 104 cells/well)

in 0.5 mL of media and infected at a multiplicity ofinfection (MOI) of 1.5 in triplicate. LY294002 was added6 hours postinfection (pi) and titers determined by plaqueassay on Vero cells.

Cell susceptibility assays and Chou–Talalay analysisCells were seeded into 96-well plates (5,000 cells/well),

and 3.5 days after infection or 3 days after drug treatmentMTS assays (Promega) were carried out according to man-ufacturer’s instructions. For Chou–Talalay analysis (21),

Translational Relevance

Glioblastoma (GBM) is an invariably lethal braintumor. Glioblastoma stem cells (GSC) are thought tobe important in disease progression, recurrence, andresistance to therapy, providing an important target forthe development of new therapies. Oncolytic herpessimplex viruses (oHSV) hold promise for GBM treat-ment; however, evidence for clinical efficacy remainselusive. One strategy to enhance the efficacy of US3-deleted oHSV is combination with phosphoinositide-3-kinase (PI3K)/Akt pathway inhibitors. This study showsthat oHSV-MG18L replicates well in GSCs and is effi-cacious in mice bearing GSC-derived brain tumors, yetnonneuropathogenic in mice. Notably, MG18L syner-gizes with PI3K/Akt inhibitors in killing GSCs and theirintracerebral tumors. This study is the first to showeffective targeting of cancer stem cells by the combina-tion of oHSV and small molecule inhibitors. Synergywith MG18L may be utilized to increase efficacy ofPI3K/Akt inhibitors. These data provide support fortranslation of this novel strategy in GBM patients.

oHSV and PI3K/Akt Inhibitors Synergize in Glioblastoma

www.aacrjournals.org Clin Cancer Res; 17(11) June 1, 2011 3687




experiments were carried out as described (22). Dose–response curves and 50% effective dose values (ED50) wereobtained, and fixed ratios of drug and virus and mutuallyexclusive equations used to determine combination indices(CI). Briefly, combined dose–response curves were fitted toChou–Talalay lines (21), which are derived from the law ofmass action and described by the equation: log(fa/fu) ¼ mlog(D) � m log(Dm), in which fa is the fraction affected, fuis the fraction unaffected, D is the dose, Dm is the median-effect dose, and m is the coefficient signifying the shape ofthe dose–response curve. The CI values were calculatedusing the equation: CI ¼ (D1/Dx1) þ (D2/Dx2) þ (D1)(D2)/[(Dx1) (Dx2)], where Dx1 and Dx2 are the inhibitorand virus doses, respectively, that are required to achieve aparticular fa, and D1 and D2 are the doses of the 2 agents(combined treatment) required for achieving the same fa.CI < 1, CI¼ 1, and CI > 1 indicate synergistic, additive, andantagonistic interactions, respectively.

ImmunoblotsCells were treated with virus at MOI of 1.5, collected at

indicated times after infection, and cell pellets lysed in RIPA(radioimmunoprecipitation assay) buffer (Boston Biopro-ducts) with a cocktail of protease and phosphatase inhi-bitors (Roche). A total of 12.5 mg of protein was separatedby 10% SDS-PAGE and transferred to PVDF (polyvinyli-dene difluoride) membranes by electroblotting. Afterblocking with 5% nonfat dry milk in TBS-Tween 20,membranes were incubated at 4�C overnight with thefollowing antibodies (1:1,000): Akt, phosphorylated Akt(p-Akt; Ser473), phosphorylated Akt (Thr308), cleavedPARP (all from Cell Signaling Technology), ICP4 (USBio-logical), or actin (Sigma), followed by incubation withappropriate HRP (horseradish peroxidase)-conjugated goatanti-rabbit or anti-mouse secondary antibodies (1:5,000;Promega) for 1 hour at room temperature. Protein–anti-body complexes were visualized using ECL (AmershamBioscience).

Assay of caspase activityCells seeded into 96-well plates (5,000 cells/well) in

triplicate were infected, LY294002 (20 mmol/L) or vehiclealone added 10 hours later, and caspase-3/7 activity eval-uated 16 hours after infection using the Caspase-Glo 3/7Assay Kit (Promega) according to the manufacturer’sinstruction.

Animal experimentsAll in vivo procedures were approved by the Subcom-

mittee on Research Animal Care at Massachusetts GeneralHospital. For safety evaluation, female A/J mice, 6 weeksof age (NCI, Frederick, MD), were stereotactically inocu-lated (right striatum, 2.5-mm lateral from Bregma and2.5-mm deep) with virus in 3 mL of virus buffer (150mmol/L NaCl, 20 mmol/L Tris, pH 7.5) and euthanizedwhen moribund. For efficacy studies, female athymicmice, 6 to 8 weeks of age (NCI) were stereotacticallyimplanted (right striatum, 2.5-mm lateral from Bregma

and 2.5-mm deep) with dissociated BT74 cells (1� 105 in3 mL). On day 8, randomly grouped mice were treated byintratumoral injection of MG18L or virus buffer in 3 mL,followed 12 hours later with intraperitoneal injection ofLY294002 or solvent daily for 5 days. For mice surviving100 days, the absence of tumor tissue was macroscopi-cally confirmed. For histologic studies, mice (28 daysafter tumor implantation) were treated with mock,MG18L, and/or LY294002 (days 28 and 29). Upon sacri-fice brains were removed, fixed in 4% paraformaldehyde,and frozen sections subjected to X-gal and hematoxylinstaining or immunocytochemistry with antibodiesagainst human specific nuclei (Millipore), cleaved cas-pase-3 (Cell Signaling Technology), nestin (Santa CruzBiotech), or b-gal (mouse from Sigma or rabbit fromMillipore), followed by incubation with appropriate FITC(fluorescein isothiocyanate)- and Cy3-conjugated sec-ondary antibodies (Jackson ImmunoResearch) and DAPI(40,6-diamidino-2-phenylindole).

StatisticsComparisons of data in viral replication and caspase-3/7

assays were carried out using a 2-tailed Student’s t test(unpaired). Survival was analysed by Kaplan–Meier curveswith comparisons by log-rank test. p values less than 0.05were considered statistically significant.

Results

Central nervous system safety and selectivity ofMG18L

MG18L is a new oHSV generated for GBM therapy thatcontains a deletion of the US3 gene and a LacZ insertioninactivating ICP6 (UL39; Fig. 1A). As isogenic controls, weused FD6, containing only the same ICP6 insertion instrain F, and R7041 with only the US3 deletion. Similar toG207, replication of MG18L was highly compromised inhuman astrocytes compared with parental R7041 andFD6. In U87 human glioma cells, MG18L replicated toa greater extent than G207 and somewhat less than R7041and FD6 (Fig. 1B). For oHSV to be translatable to theclinic for delivery in the brain, it is critical that it is notneuropathogenic. To examine this, highly HSV-1 suscep-tible A/J mice were inoculated intracerebrally and eval-uated for neurologic symptoms. All mice inoculated withas little as 4 � 102 plaque-forming units (pfu) of wild-type HSV-1 (strain F) died, whereas all mice inoculatedwith 4 � 106 pfu of MG18L (the highest dose obtainablein 3 mL) or FD6 survived. In contrast, this dose of R7041killed 90% of mice (Table 1). We further quantifiedneurologic deficits using a scoring scale for morbidity,with 4 of 6 mice inoculated with FD6 (4 � 106 pfu)showing moderate symptoms, whereas 2 of 8 mice inocu-lated with MG18L at this dose exhibited only mild tran-sient symptoms (Fig. 1C). At a 10-fold lower dose, only 2of 6 R7041-inoculated mice did not show deficits(Fig. 1C). Thus, we conclude that MG18L is safe andselective for glioma cells.

Kanai et al.

Clin Cancer Res; 17(11) June 1, 2011 Clinical Cancer Research3688




Effects of MG18L against human GSCs in vitroAll GSCs tested were susceptible to MG18L replication,

with virus yield being similar to R7041 in GBM8 andGBM13 and 4- to 5-fold less in GBM4 and BT74, whereasG207 does not replicate (Fig. 2A). One activity of US3protein is to inhibit Akt phosphorylation at Ser473 inducedby HSV infection (17, 18). Phosphorylation of Akt is

induced by FD6 and G207 in GBM4 and BT74, respectively,even though G207 does not replicate, as illustrated byshutoff of immediate early ICP4 expression (Fig. 2B). InMG18L-infected GSCs, p-Akt (Ser473 but not Thr308)levels are further increased and remain stable or continueto increase with time (Fig. 2B). The in vitro efficacy ofMG18L was compared with FD6 and G47D in GSCs. FD6was most effective at killing GSCs, whereas MG18L wassimilar to G47D, except in GBM8 (Fig. 3A). The virus yieldsfor G47D and FD6 were previously reported (15). This wassimilar in glioma cell lines U87 and T98G (SupplementaryFig. S2B).

Human GSCs exhibit variable sensitivities to PI3K/Aktpathway inhibitors in vitro

The PI3K/Akt signaling pathway, commonly activated incancer, is one of the most active targets for drug develop-ment. As a prelude to examining the interaction betweenMG18L and PI3K/Akt pathway inhibitors, we determinedthe GSC sensitivity to inhibitors of PI3K (GDC-0941), Akt(triciribine), mTOR/PI3K (BEZ235), and nonspecific PI3K(LY294002; ref. 23). GBM13 was the least sensitive to all 4inhibitors, followed by BT74, whereas GBM4 and GBM8were quite sensitive (Fig. 3B). Triciribine (�250 mmol/L)had no effect on GBM13 viability, so its ED50 was notobtainable. Both U87 and T98G exhibited a similar rangeof inhibitor sensitivities as BT74 (Supplementary Table S1).

Table 1. Safety evaluation of oHSV after intra-cerebral inoculation in A/J mice

Dose, pfu Virus

Strain F(wild-type)

R7041 FD6 MG18L

4 � 106 NT 1/10 6/6 8/82.5 � 106 NT NT NT 3/34 � 105 NT 3/6 NT NT4 � 104 NT 6/6 NT NT4 � 103 0/3 NT NT NT4 � 102 0/3 NT NT NT

NOTE: Mice were observed for 28 days and the number ofsurvivors/total indicated.Abbreviation: NT, not tested.

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Figure 1. Characterization of MG18L. A, schematic of MG18L, with E. coli lacZ coding sequence inserted into the UL39 gene of R7041, which containsa deletion at US3. Boxes represent inverted repeat sequences flanking the long (UL) and short (US) unique sequences (B, BamHI; G, BglII; X, XhoI).B, replication of HSV-1 strain F and its derivatives in human astrocytes (HA; left) and U87 (right). Cells were infected at MOI of 1.5, 24 hours later, cellsand media were harvested and viral titers were determined. Dotted lines indicate the input amount of virus. *, P < 0.05. C, morbidity caused by intracerebralinoculation of HSV mutants evaluated with the following scoring scale; general appearance, spontaneous activity, reaction to external stimuli orneurologic deficits, each scoring from 1 to 4 (with 1 severely impaired to 4 normal) for a total score of 3 to 12. Time course of morbidities caused byR7041 (4 � 105 pfu; left), FD6 (4 � 106 pfu; middle), and MG18L (4 � 106 pfu; right) indicated for each mouse (symbols).






MG18L synergizes with PI3K/Akt pathway inhibitorsto inhibit human GSCs in vitro

One of the rationales for the construction of MG18L wasits postulated ability to sensitize cancer cells to PI3K/Aktpathway inhibitors. MG18L and LY294002 synergized inkilling 3 of the 4 GSCs, whereas the interaction in humanastrocytes was antagonistic to additive (Fig. 3C–G). Synergywas dependent on the US3 deletion, as the interactions ofFD6 and G47D (US3

þ oHSV) with LY294002 were mostlyadditive or antagonistic (Fig. 3C–F). Similarly, the interac-tions in glioma cell lines (U87, T98G) were synergistic onlyfor US3

� R7041 and MG18L (Supplementary Fig. S3A).Synergy in U87 cells was not affected by the order ofaddition (Supplementary Fig. S3A). As LY294004 has abroad inhibitor profile across many PI3Ks and proteinkinases (24), we examined the interactions of MG18L withselective PI3K/Akt pathway inhibitors that have been in

clinical trial. Both triciribine and GDC-0941 synergizedwith MG18L to kill GBM4 and BT74 (Fig. 3H and I),whereas the dual PI3K/mTOR inhibitor BEZ235 onlysynergized in GBM13 (Fig. 3J). Because GBM13 was resis-tant to triciribine (Fig. 3B), we were unable to carry out asynergy analysis; however, a nontoxic dose of triciribine(30 mmol/L) enhanced killing byMG18L (data not shown).The interactions were similar in the glioma cells lines, withBEZ235 only strongly synergistic in T98G (SupplementaryFig. S3B–D).

The combination of MG18L and LY294002 activateapoptotic pathways in glioma cells in vitro

Treatment of BT74 or U87 cells with LY294002 didnot increase MG18L replication and virus yield (Supple-mentary Fig. S4), indicating that this is not the mechan-ism underlying synergy. Because US3 blocks apoptosis in

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Figure 2. oHSV replication in GSCs and activation of Akt. GBM4 (top), GBM8 (middle), GBM13 (middle), and BT74 (bottom) were infected with G207,FD6, or MG18L at MOI of 1.5. A, single-step virus growth analysis. cells and media were collected and virus yields determined. B, MG18L infectionstrongly activates Akt in GSCs. Collected cells were processed for Western blotting with antibodies to ICP4 (immediate-early virus gene expression),p-Akt (Ser473 and Thr308), and total Akt.

Kanai et al.





HSV-infected cells (16, 25), we hypothesized that thecombination of MG18L and PI3K/Akt pathway inhibitorsenhanced apoptosis in glioma cells, contributing to theobserved synergy. Caspase-3 and -7 are common effectorcaspases of both the intrinsic and extrinsic apoptoticpathways. GSCs and U87 infected with FD6 or MG18Lshowed increased levels of caspase-3/7 activity, withMG18L being significantly more effective than FD6

(Fig. 4A–D and Supplementary Fig. S5B). Treatment ofall GSCs and U87 cells, except GBM8 where synergistickilling was not observed, with MG18L and LY294002showed significantly higher activities of caspase-3/7,compared with treatment with either alone (Fig. 4A–Dand Supplementary Fig. S5B). The combination of FD6and LY294002 did not produce an increase in caspaseactivity (Fig. 4A–D and Supplementary Fig. S5B), further

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Figure 3. Effects of oHSV or PI3K/Akt pathway inhibitors on GSC viability and the interaction of the combination. A, GSCs and human astrocytes wereinfected with G47D, FD6, or MG18L at different MOIs and 3.5 days later, viability was determined, dose–response curves obtained, and ED50 dosesdetermined. B, cell viability was determined 3 days after adding inhibitor. The ED50 dose for each GSC was normalized to the ED50 dose for GBM4 (13.2, 27.1,0.57, and 0.034 mmol/L for LY294002, triciribine, GDC-0941, and BEZ235, respectively). C–J, interactions were analyzed by the median effect method ofChou–Talalay, with data presented as fraction affected–combination index plots. CI < 1,¼ 1, > 1 represent synergistic, additive, and antagonistic interactions,respectively. C–G, interactions between oHSV (G47D, FD6, and MG18L) and LY294002 on proliferation of GBM4 (C), GBM8 (D), GBM13 (E), BT74 (F),and normal human astrocytes (G). H–J, interactions between MG18L and PI3K/Akt pathway inhibitors; triciribine (H), GDC-0941 (I), and BEZ235 (J).






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Figure 4. Combination of MG18Land LY294002 induces caspase-3/7 activation and PARP cleavagein GSCs. A–D, GSCs wereinfected with FD6 or MG18L atMOI of 1.5, or mock, and 14 hourslater, cells were treated with orwithout LY294002 (LY; 20 mmol/L),and the activities of caspase-3and -7 were evaluated (RLU;relative luminescence units) 20hours after infection. A, BT74; B,GBM4; C, GBM8; D, GBM13. E–H,GSCs were treated as in A to D,except cells were collected andprocessed for Western blottingwith antibodies to cleaved PARP(apoptosis marker), p-Akt, andtotal Akt. E, BT74; F, GBM4; G,GBM8; H, GBM13. I, synergybetween MG18L and LY294002 inBT74 was impaired by the pan-caspase inhibitor Z-VAD-FMK(50 mmol/L). The values for MG18Land LY204002 are the same as inFigure 3F.

Kanai et al.





showing the involvement of the lack of US3. As anadditional readout for apoptosis, we examined cleavedPARP. Consistent with the results from the caspase-3/7activity assay, an increased level of cleaved PARP wasobserved with the combination, except for GBM8(Fig. 4E–H and Supplementary Fig. S5A). In all cells,including GBM8, LY294002 treatment inhibited Akt acti-vation (Fig. 4E–H and Supplementary Fig. S5A). Theseresults support the view that synergy is because ofincreased apoptosis. Blocking caspase activation with apan-caspase inhibitor, Z-VAD-FMK, before treatmentwith MG18L and LY294002, impaired synergy in BT74,GBM4, and U87 cells (Fig. 4I and Supplementary Fig. S5Cand D).

MG18L treatment of human GSC–derivedintracerebral tumorsTo evaluate the in vivo efficacy of MG18L, we used BT74-

derived intracerebral tumors. Histopathologically, thisGSC forms aggressive tumors with high levels of vascularityand intratumoral hemorrhage (Fig. 5A, top; ref. 15), ahallmark of GBM in humans. MG18L efficiently infectedand spread within the tumor, as illustrated by the extensiveX-gal staining at 36 hours postinfection, which was moreextensive than at 12 hours (Fig. 5A, bottom). Sections weredoubly immunostained with antibodies against humannuclear antigen (identifies BT74 cells) and b-galactosidase(identifies MG18L-infected cells), confirming MG18Lspread within the tumor tissue (Fig. 5B). Most of the cellsin the tumors stained positive for nestin and these weresusceptible to MG18L infection (Supplementary Fig. S6).Treatment of mice bearing established BT74 tumors with asingle injection of MG18L significantly extended theirsurvival (Fig. 5C). Median survival increased from 32.5days for mock to 41 days with MG18L, with 1 animalsurviving 100 days.

LY294002 enhances the therapeutic efficacy of MG18Lin vivoLY294002 treatment alone of BT74 tumor–bearing mice

was only modestly effective, with a prolongation ofmediansurvival by 5.5 days (Fig. 5C). Combination with MG18Lwas much more efficacious than either treatment alone,with 50% of mice surviving long term (Fig. 5C). At 100days, when all surviving mice were sacrificed, we did notdetect tumor macroscopically. Similar results wereobtained in a U87 intracerebral tumor model (Supplemen-tary Fig. S7). Because combination treatment increasedapoptosis in vitro (Fig. 4), we examined BT74 tumor sec-tions frommice for the presence of apoptotic cells (cleavedcaspase-3 immunocytochemistry). Mock treatment did notinduce detectable cleaved caspase-3 within the tumor,whereas LY294002 induced scattered positive cells(Fig. 5D and E). In contrast, MG18L infection induced asignificant number of cleaved caspase-3–positive cells,which was further increased in combination withLY294002 (Fig. 5D and E), further supporting the hypoth-esis that apoptosis is a key mediator of improved efficacy.

Discussion

We have constructed a new oHSV vector, MG18L, con-taining a US3 deletion and ICP6 mutation that is both"safe" after intracerebral inoculation in HSV-susceptiblemice and efficacious against GSCs, warranting clinicaltranslation for GBM and possibly other tumors. The non-essential HSV US3 gene is a serine-threonine kinase withmultiple biological functions, including blocking virus andexogenously induced apoptosis (16, 26), so that its lossshould engender tumor selectivity, although this stillremains to be shown. In fact, US3 mutants in both HSV-1 and HSV-2 have been shown to have oncolytic activity(18, 27). Other US3 activities that may be pertinent includephosphorylation of histone deacetylase (HDAC) 1 andHDAC2 blocking activity (28), phosphorylation of IFN-greceptor a blocking downstream signaling (29), and inhi-biting virus-induced Akt activation (17, 18). While R7041,with a single US3 mutation, was found to be very attenu-ated for pathogenicity in the periphery (18, 30, 31), it wasnot after intracerebral inoculation (32, 33). We found thatR7041 had an LD50 (lethal dose) of about 4 � 105 pfu inA/J mice, whereas others reported LD50 values of 1.8 � 106

and 1 � 105 pfu (32, 34). Therefore, to improve safety, wecombined the US3 deletion with an ICP6 mutation thatfurther attenuates neurovirulence (Fig. 1C) and increasessensitivity to antivirals yet does not greatly reduce virusreplication in GSCs (15, 35). After intracerebral inocula-tion, MG18L exhibited a favorable safety profile that wascomparable to g34.5 deletion mutants such as G207 andG47D, which have been safely administered to GBMpatients (12, 14, 36).

MG18L replicates well in GSCs and glioma cell lines, butnot human astrocytes, and was very cytotoxic to GSCs invitro, similar to G47D but less than FD6. While the additionof the ICP6 mutation to US3-deleted oHSV reduces replica-tion in some GSCs, this is offset by decreased replication inastrocytes and improved safety. Themechanism underlyingMG18L selectivity for GSCs, as well as apoptotic pathwayfunction, remains to be determined. As expected, GSCinfection with MG18L induced higher levels of p-Akt thanFD6 infection. There are at least 10 inhibitors targeting thePI3K/Akt pathway in clinical trial including for glioma (7,23). MG18L synergized with PI3K/Akt inhibitors(LY294002, triciribine, and GDC-0941) in killing GSCs,except for GBM8, but not human astrocytes. Recently, itwas reported that increased p-Akt, in response to DNA-damaging agent doxorubicin, correlated with PI3K inhibi-tor synergy (37), possibly through a similar mechanism aswith oHSV. Synergy was dependent on the US3 deletion, asother oHSVs expressing US3 (G47D and FD6) did notsynergize (Fig. 3). In contrast, synergy did not seem tocorrelate with how susceptible cells were to the PI3K/Aktinhibitors or MG18L alone, as the ED50 values variedmorethan 10-fold, and GBM8 had very similar sensitivities asGBM4 (Fig. 3). Interestingly, the dual PI3K/mTOR inhibi-tor, BEZ235, was found to only synergize with MG18L inGBM13 and was mostly antagonistic in GBM4 and BT74.






GBM13 was much less sensitive to the PI3K/Akt pathwayinhibitors than the other GSCs and astrocytes, althoughsynergy was also observed in T98G cells, which was quitesensitive to the inhibitors alone. GBM13 is the only GSCtested that did not express PTEN (data not shown), andT98G has a PTEN mutation (38). We tried to examinesynergy with the mTOR inhibitor rapamycin but could notobtain reliable dose–response curves for analysis of

synergy. However, when a single dose of rapamycin wascombined with serial dilutions of MG18L in GBM4 orBT74, the reduction in cell viability was much less thanadditive, which may have contributed to the antagonisticinteraction with BEZ235.

PI3K inhibition with LY294002 did not affect MG18Lreplication, which could have been one mechanism forsynergy. Instead, the mechanism seems to be due to

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Figure 5. Combination therapy extends survival of mice bearing intracerebral BT74 tumors. A, top, athymic mice intracerebrally implanted with BT74were sacrificed on day 30. Shown is a representative section of the brain with untreated BT74 tumor (hematoxylin and eosin staining) illustrating intratumoralhemorrhage (arrow). Bottom, mice treated with MG18L (2 � 106 pfu) on day 28 were sacrificed 12 hours (left) and 36 hours (right) later. Sections werestained with X-gal (blue) to indicate MG18L infected cells. B, a region at the edge of the tumor from a 36 hour pi brain after immunostaining with antibodiesagainst b-gal (green; MG18L infected cells) and human nucleus (red; BT74 cells), followed by DAPI (total nuclei; scale bar ¼ 100 mm). Virus was injectedto the right of the displayed field. C, Kaplan–Meier survival curves of mice bearing BT74 tumors after treatment with intratumoral MG18L (2 � 106 pfu)or mock, and intraperitoneal LY294002 (LY; 25 mg/kg/d) or vehicle (mock, n ¼ 10; MG18L, n ¼ 10; LY294002, n ¼ 6; combination, n ¼ 6). P < 0.05(mock vs. LY294002 and LY294002 vs. MG18L þ LY294002), P < 0.0005 (mock vs. MG18L; log-rank test). D, mice with BT74 tumors treated withmock, MG18L, LY294002, or the combination were sacrificed 36 hours after MG18L injection. Brains were sectioned and tumors immunostained withantibodies to b-gal (green; MG18L) and cleaved caspase-3 (red; apoptosis) followed by DAPI (scale bars¼ 100 mm). E, the ratio of cleaved caspase-3–positivecells/total was determined in 3 randomly selected fields. *, P < 0.05; **, P < 0.01.

Kanai et al.





enhanced apoptosis in combination-treated cells. Interest-ingly, only in MG18L-infected GBM8 cells, apoptosis wasnot increased by LY294002. Thus, synergy with LY294002could be impaired or abrogated in the presence of the pan-caspase inhibitor Z-VAD-FMK in BT74, GBM4, and U87cells (Supplementary Figs. S4 and 5). This supports theview that apoptosis induced by HSV infection that is notblocked by US3 is suppressed by activated Akt, balancingcompeting US3 altered pathways and making the cells verysensitive to inhibition of Akt signaling. Inhibition of thePI3K/Akt pathway in glioma has been reported to eitherinduce apoptosis or autophagy (39–42), and sensitize cellsto chemotherapy-induced apoptosis (43). LY294002 treat-ment of GSCs significantly induced apoptosis in GBM4 andGBM8 but not GBM13 or BT74. The range of interactionsobserved in the different GSCs illustrates the complexity ofthe PI3K/Akt pathway in GBM, with its multiple feedbackloops overlaid with genetic alterations at different nodes inthe pathway, as well as potential interactions with the MEK(MAP/ERK kinase) pathway (44). Further genetic analysisof the GSCs may identify alterations underlying sensitivityto inhibitors at different steps in the PI3K/Akt pathway andprovide insights for future stratification of patients in anyclinical trials with inhibitors in this pathway.As a proof-of-concept study, we examined the combina-

tion of LY294002 with MG18L for in vivo therapy in micebearing BT74 tumors. Combination treatment significantlyprolonged the survival of mice, with 50% long-term survi-vors, compared with either agent alone. Synergy in vitroresulted in a 6.5-fold reduction in LY294002 dose at theED50, to a more obtainable dose in vivo. While synergy invitrowas not affected by the temporal order of addition, forthis in vivo experiment we administered MG18L prior toLY294002 to allow for induction of Akt activation byMG18L infection, followed by Akt inhibition by moleculartargeting drugs. It will be important for clinical translationto further characterize the effect of administration schedule

on efficacy with clinically relevant inhibitors. Although, wefound that the combination treatment increased apoptosisboth in vitro (Fig. 4) and in vivo (Fig. 5D and E), there maybe additional factors contributing to the enhanced efficacyof the combination in vivo. For example, the antiangiogenicproperties of PI3K/Akt/mTOR inhibitors (45, 46) anddisruption of tumor interstitial space produced byenhanced apoptosis may have helped the spread ofMG18L in the tumor tissue (47). In summary, MG18L iseffective in killing human glioma cells including GSCs, andthe therapeutic efficacy can be enhanced by combinationwith PI3K/Akt pathway inhibitors. This strategy of using anoncolytic virus to alter a cancer pathway so that it sensitizescancer cells to small molecule inhibitors that molecularlytarget these pathways is a novel one that should be applic-able to other oHSV mutants, signaling pathways, andcancers.

Disclosure of Potential Conflicts of Interest

S.D. Rabkin was a consultant/advisory board member of MediGene.

Acknowledgments

The authors thank Drs. Bernard Roizman (University of Chicago) forR7041 and Sandra K. Weller (University of Connecticut Health Center) forpKX2-bG3, Cecile Zaupa for assistance with virus constructions, and Mat-thew Gardner for preliminary synergy experiments.

Grant Support

The study was supported by NIH grant NS-032677 (R.L. Martuza) andDepartment of Defense grant W81XWH-07-1-0359 (S.D. Rabkin).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 24, 2010; revised March 30, 2011; accepted April 7,2011; published OnlineFirst April 19, 2011.

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2011;17:3686-3696. Published OnlineFirst April 19, 2011.Clin Cancer Res Ryuichi Kanai, Hiroaki Wakimoto, Robert L. Martuza, et al. Glioblastoma Stem CellsPhosphoinositide 3-kinase/Akt Pathway Inhibitors to Target A Novel Oncolytic Herpes Simplex Virus that Synergizes with

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